During the CAA webinar this afternoon (this covered three of the current funded research projects - very interesting, btw), there was a slide that mentioned a new paper accepted for publication today by Brain Behavior and Immunity. It sounds like it will be epublished in the next week - I couldn't find the paper or abstract online just now on a quick search, but it should show up soon.

Gordon Broderick is lead author, with Klimas and Fletcher also involved, on a study using a Klimas research cohort. (He is one of the information network gurus from British Columbia involved in a lot of CFS studies in recent years - I keep hearing great things about him, and think what he brings is highly valuable to a complicated area like CFS.)

My notes are a bit rough, but I think Suzanne Vernon said that the paper shows processes consistent with viral expression in latent infection, that wouldn't have been uncovered with a single marker.

I don't know if the paper discusses how these processes may distinguish CFS patients from healthy cohort.

There were some interesting charts / graphs during the webinar - well worth looking at when they're available online, along with the discussion.

there was a slide that mentioned a new paper accepted for publication today by Brain Behavior and Immunity. It sounds like it will be epublished in the next week - I couldn't find the paper or abstract online just now on a quick search, but it should show up soon.

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Thanks for the tip-off, will you post here once published? The Dubbo studies suggested that brain/cytokine interactions might be central to CFS.

Thanks for the tip-off, will you post here once published? The Dubbo studies suggested that brain/cytokine interactions might be central to CFS.

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Ocean, I will try to check later in the week - the next couple of days I'm likely to be off-line. If it comes out before then someone else will most likely pick up on it but maybe on another thread. It sounds very interesting.

Thanks for the tip-off, will you post here once published? The Dubbo studies suggested that brain/cytokine interactions might be central to CFS.

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Sorry to be awkward but I don't think any of the Dubbo studies really showed that. Lloyd tried to claim that because in one paper, there was no evidence of ongoing immunological problems, it must be to do with the brain. That's how I recall it.

a Department of Medicine, University of Alberta, Edmonton, Alberta, Canada

b The CFIDS Association of America, Charlotte, NC, USA

c Miami Veterans Affairs Medical Center, Miami, FL, USA

d Department of Medicine, University of Miami, Miami, FL, USA
Received 1 February 2010;
revised 21 April 2010;
accepted 28 April 2010.
Available online 4 May 2010.

Abstract

Chronic Fatigue Syndrome (CFS) is a complex illness affecting 4 million Americans for which no characteristic lesion has been identified. Instead of searching for a deficiency in any single marker, we propose that CFS is associated with a profound imbalance in the regulation of immune function forcing a departure from standard preprogrammed responses. To identify these imbalances we apply network analysis to the co-expression of 16 cytokines in CFS subjects and healthy controls. Concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12, 13, 15, 17 and 23, IFN-γ, lymphotoxin-α (LT-α) and TNF-α were measured in the plasma of 40 female CFS and 59 case-matched controls. Cytokine co-expression networks were constructed from the pair-wise mutual information (MI) patterns found within each subject group. These networks differed in topology significantly more than expected by chance with the CFS network being more hub-like in design. Analysis of local modularity isolated statistically distinct cytokine communities recognizable as pre-programmed immune functional components. These showed highly attenuated Th1 and Th17 immune responses in CFS. High Th2 marker expression but weak interaction patterns pointed to an established Th2 inflammatory milieu. Similarly, altered associations in CFS provided indirect evidence of diminished NK cell responsiveness to IL-12 and LTα stimulus. These observations are consistent with several processes active in latent viral infection and would not have been uncovered by assessing marker expression alone. Furthermore this analysis identifies key sub-networks such as IL-2:IFNγ:TNFα that might be targeted in restoring normal immune function.

This I find interesting: Furthermore this analysis identifies key sub-networks such as IL-2:IFNγ:TNFα that might be targeted in restoring normal immune function.

I have not seen interferon gamma mentioned before. The immunomodelator I use is supposed to stimulate interferon gamma production and being helpfull in restoring normal Th1/Th2

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL- 5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL- 2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.

Sorry to be awkward but I don't think any of the Dubbo studies really showed that. Lloyd tried to claim that because in one paper, there was no evidence of ongoing immunological problems, it must be to do with the brain. That's how I recall it.

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Later analysis showed that during onset cytokine levels were correlated with symptom severity and patients with both had a significantly higher risk of developing CFS than people with viral infection with low cytokines; ie high cytokine levels caused increased symptoms and it was the highly symptomatic people who went onto develop CFS.

Their theory I think was that high cytokine levels early in the process somehow effected brain functioning.

After that early period no immunological abnormalities were seen in the people who remained ill.

Later analysis showed that during onset cytokine levels were correlated with symptom severity and patients with both had a significantly higher risk of developing CFS than people with viral infection with low cytokines; ie high cytokine levels caused increased symptoms and it was the highly symptomatic people who went onto develop CFS.

Their theory I think was that high cytokine levels early in the process somehow effected brain functioning.

cytokines do affect brain functoning at
number of levels.Interferon alpha for example interferes with HPA functioning. i
would like to know if the patients experienced PEM.My objective is not to be critical.I ask because clinical depression is also associated with abnormal cytokine patterns.Given that the reeves definition and the majority of the fukuda presentations is incapable of distinguishing between Myalgic Encephalopathy(using Dr Klimas,s new term) and clinical depression the possibility that this pattern is a result of depression remains.If they have PEM(If DR KLIMAS HAS BEEN INVOLVED IN DIAGNOSIS I<M SURE THAT THEY HAVE) then the study should explicitly say so.This would be in line with the fukuda research guidelines which emphasise that merely stating that the patients fulfilled diagnostic critiria is insufficient.The patient characteristics need to be clearly stated so that meaningful comparison of research can be achieved.this research is very very good but it is such a shame that a third group was not added with people with clinical depression.If it had been then we for the first time we would have known that this cytokine signature was specific to Myalgic encephalopathy and different to that seen in depressed people.We would have also known whether Reeves could diagnose anything objective at all.I wonder if Dr Klimas could repeat the study to answer these questions.Perhaps she is indeed planning to

Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal

Received 8 May 2009; Accepted 23 June 2009

Academic Editor: Hari Manev

Copyright 2009 Susana Roque et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

An increasing body of evidence implicates proinflammatory cytokines in psychiatric disorders, namely, in depression. Of notice, recent studies showed that anti-inflammatory cytokines, such as IL-10, also modulate depressive-like behavior. In this article, we propose that the anti-inflammatory cytokine IL-10 is a putative link between two of the most widely reported phenomenon observed in depressed patients: the disruption of the hypothalamic-pituitary-adrenal axis and the imbalanced production of cytokines. If so, IL-10 might represent a novel target for antidepressant therapy.

1. Introduction

The establishment of a bidirectional interaction between the immune and central nervous systems is one of the most remarkable findings of the last decades. These two systems accomplish extremely different functions, but a growing body of evidence shows that both share common mediators, including cytokines and their receptors. Therefore, the altered expression of these molecules, triggered by one of the systems, might influence the other in a reciprocal way [1]. As a consequence, a disruption in this cross-talk has been causally implicated in neuropathological features associated with several psychiatric disorders, in particular depression [2].

Major depression is a global public-health problem and a leading cause of disability worldwide, representing the fourth contributor to the global burden of disease in 2000 (in terms of DALYs-Disability Adjusted Life Years) [3]. Depression is a heterogeneous disorder with a variable set of symptoms, diverse disease courses, and inconsistent responses to treatment. The etiology of depression is still controversial, and several theories have emerged to explain it. Taking into account the communication between the immune and central nervous systems, Smith, in the early 1990s, proposed a role for cytokines in depression [4]. The “cytokine theory of depression”, that has been widely studied in the last two decades, proposes that enhanced production of proinflammatory cytokines is associated with the pathogenesis of depression. Indeed, several studies show a significant increase in the production of proinflammatory cytokines (namely, IL-6, IL-1β, IFN-γ, and TNF) among depressed patients [5–8]. Moreover, immunotherapy based on the administration of IFN-α and IL-2, which is frequently used as part of the treatment against chronic hepatitis C and certain cancers, has been associated with depressed mood and symptoms of cognitive impairment and fatigue [8–10]. The fact that the symptoms associated with depression disappear almost immediately after termination of cytokine administration supports a causal role for cytokines in the disease; moreover, these symptoms can also be relieved by the administration of antidepressant drugs [11].

The “cytokine theory of depression” has also been supported by a large set of results in animals models. Research using these models demonstrated that alteration in the proinflammatory cytokine milieu can lead to behavioral changes overlapping with those found in depressed patients, including anhedonia, decreased activity, cognitive dysfunction, and altered sleep patterns [12]. In further support of this link, mice exposed to a chronic mild stress (CMS) protocol, which induces symptoms of depressive-like behavior, show increased levels of IL-1 in the hippocampus [13]. Furthermore, the depressive-like behavior observed after CMS can be mimicked by chronic administration of IL-1 [13] and, when mice lacking the expression of IL-1 receptor are exposed to the same CMS paradigm, the behavioral changes do not occur [13]. Interestingly, mice unable to express the receptor for TNF, one of the cytokines more consistently upregulated in depression, show decreased immobility in the forced swim test (FST), that is, decreased signs of depressive-like behavior in a test that assesses learned helplessness and is considered one of the gold standard tests to evaluate depression in rodents [14]. These observations suggest that TNF activation also mediates depressive-like behavior in mice.

Although most studies on the “cytokine theory of depression” are centered on increased levels of proinflammatory cytokines, the role of anti-inflammatory cytokines has been recently analyzed. Of notice, IL-10, one of the most important anti-inflammatory cytokines, proved to be relevant in depression [15]. Specifically, mice lacking the expression of IL-10 (IL-10KO) show a decreased latency to immobility and longer immobilization time than wild-type (WT) mice in the FST [15]. These results demonstrate that IL-10KO mice display increased helplessness, a recognized sign of depressive-like behavior in rodents. Remarkably, administration of IL-10 is able to revert the depressive-like phenotype observed in the IL-10KO animals [15]. In further support of a role for IL-10 in depression, transgenic mice overexpressing this cytokine show a decreased depressive-like behavior in the FST in comparison with WT animals [15]. In addition to the increased helplessness in mice that lack the expression of IL-10, other studies also reported that modulation of IL-10 impacts on psychophysiological alterations frequently observed in depression. Among these is the impairment in sleep behavior [16]. Interestingly, IL-10KO mice show alterations in the sleep pattern, and the exogenous administration of IL-10 modulates sleep behavior [17–19].

Another feature commonly associated with depression is altered pain perception [20]. Tu and coworkers described, using the IL-10KO mouse model, that the absence of this anti-inflammatory cytokine is associated with decreased nociception (i.e., the ability to sense painful stimuli). This study showed that IL-10KO mice have an increased latency time to paw licking (the time that mice spend to avoid a heat stimulus by moving away and lick the paw) compared with WT mice, and this result was confirmed by the blockage of IL-10 in WT mice [21]. This constitutes an additional evidence that IL-10 can also be involved in the common biological pathways shared by pain and depression.

The studies described above show that modulation of IL-10 impacts on several symptoms associated with depression, namely, helplessness, sleep disturbances, and pain perception. Noticeable, administration of IL-10 can modulate these symptoms which suggests a putative antidepressant effect of the anti-inflammatory cytokine IL-10. Of relevance for this hypothesis is the observation, in depressed patients and also in animal models of depression, of increased IL-10 levels after treatment with several classes of antidepressants [22–25].

Although it is becoming clear that modulation of IL-10 can lead to changes in the normal behavior of humans and animals, the mechanisms behind these alterations remain to be elucidated. One might envisage several pathways through which IL-10 influences behavior. One of the most obvious hypotheses is the impact this anti-inflammatory cytokine may have on the levels of proinflammatory cytokines. In fact, it is admissible that the absence of IL-10, in IL-10KO mice, leads to an increased production of proinflammatory cytokines that could, in accordance with the “cytokine theory of depression”, trigger depression. This mechanism could also explain the abrogation of the depressive-like behavior in IL-10KO mice upon IL-10 administration [15], as IL-10 is known to inhibit the expression of proinflammatory cytokines [26]. However, this possibility is not supported by our results, as we were unable to detect two of the most relevant proinflammatory cytokines (IFN-γ and TNF) in the serum of IL-10KO mice [15]. More studies are certainly necessary to clearly define how the lack of IL-10 impacts on the production of proinflammatory cytokines as it is possible that subtle (below currently detection limits) changes in the concentration of these cytokines are present in the serum of IL-10KO mice, and/or that the absence of IL-10 induces the upregulation of proinflammatory cytokines in specific brain regions, which may not be reflected in the serum.

The observations that systemic IL-10 administration to WT animals induces alterations in their normal behavior (e.g., increased motor activity and abnormal exploratory patterns) [27] and that the same protocol reverts the depressive-like behavior observed in IL-10KO mice [15] indicate that peripheral IL-10 has an effect in the central nervous system. However, it is not clear how this cytokine acts within the central nervous system particularly since peripheral IL-10 does not seem to cross the intact blood-brain barrier, at least in detectable amounts [28]. It is possible that IL-10, like other cytokines, acts directly in the brain through regions devoided of blood-brain barrier, such as the circumventricular organs and the choroid plexus, or by activating vagal afferent fibers that can transmit cytokine signals to specific brain nuclei [5]. Moreover, IL-10 has been detected in normal human, rat, and mouse brains [29]. Possible sites of expression are the endothelial cells of the brain capillaries that irrigate the brain, the choroid plexus (from where it would be secreted into the cerebrospinal fluid), and also microglia and astrocytes [29–31]. Of interest, a recent study showed that several immune mediators are produced by the choroid plexus in response to systemic inflammatory stimuli [32]. In addition, the IL-10 receptor has been indentified in microglia, astrocytes [29, 30], and oligodendrocytes [29, 30, 33]; probably because of a general distribution, Ward and coworkers found expression of the receptor in all five brain regions they analyzed (cortex, cerebellum, hippocampus, hypothalamus, and pituitary) [34].

A direct action of IL-10 within the central nervous system might be mediated by its role in regulating cell survival. IL-10 has been shown to prevent cell death of glial cells [33, 35, 36] and to increase survival of cerebellar neurons [37]. Since an increase in neuronal apoptosis in the hippocampus has been associated with depression in animal models [38], this putative role of IL-10 in increasing neuronal survival should be investigated as a potential mechanisms of action in preventing depressive-like behavior.

The observation that IL-10KO mice show an increase in the adrenal and a decrease in the thymus relative weights [15] offers another alternative mechanism for the action of IL-10 in depression: the modulation of the hypothalamic-pituitary-adrenal (HPA) axis. Indeed, it was shown that, even in basal conditions, IL-10KO mice have higher levels of corticosterone and that in the presence of a stressor the increase of this hormone is even more accentuated than in WT mice [39]. These findings strongly suggest that IL-10 may have a regulatory effect in the HPA axis. One of the most consistent neurobiological alterations in depressed subjects is the hyperactivity of the HPA axis which is associated with impaired HPA axis glucocorticoid feedback sensitivity (glucocorticoid resistance) [40]. Thus, it is plausible that IL-10 modulation of depressive-like behavior is exerted through regulation of the HPA axis. In accordance, IL-10 is able to suppress, in a dose dependent manner, adrenocorticotropic hormone-(ACTH-) induced steroid production in adrenal cells [41]. This effect appears to be exerted through downregulation of enzymes responsible for the biosynthetic pathway of corticosterone [21, 41]. Interestingly, IL-10 receptor was shown to be expressed in the zona fasciculata (the region responsible for the production of glucocorticoids) of the mouse adrenal gland [41]. Accordingly, in vivo studies demonstrated that, under basal conditions, IL-10KO mice have clear signs of HPA axis activation, such as increased adrenal glands, decreased thymus relative weight, and higher levels of corticosterone [15, 39]. These findings strongly suggest that IL-10 exerts a negative regulation on corticosterone production by the adrenal gland [41]. A microarray analysis of adrenal, pituitary, and hypothalamic neural cells treated with IL-10 clearly showed that this anti-inflammatory cytokine plays a pivotal role in the regulation of several genes of the HPA axis [42]. Of notice, murine pituitary cells were the first identified nonimmune-related sources of IL-10 [43], and the presence of IL-10 was also found in human pituitary [44]. Paradoxically, in the hypothalamus and pituitary, IL-10 seems to have the same effect already described for some proinflammatory cytokines [45], which is a positive regulation of corticotrophin releasing factor (CRF) and ACTH production, respectively [43, 46]. It should be noticed that while the studies in the hypothalamus and pituitary were only performed in vitro, the inhibitory action of IL-10 in corticosterone production by the adrenal gland is a more consistent result as it is based on in vitro and in vivo studies. Taking into account that depression is often associated with hyperactivity of the HPA axis [40], we hypothesize that IL-10 has a pivotal role in the modulation of the HPA axis homeostasis, which is likely to have an impact on the etiology of depression.

Increases in glucocorticoid levels can occur daily in response to several factors. This glucocorticoid increase leads to an enhanced production of IL-10 [47] which, in normal situations, together with glucocorticoids, inhibits the activity of the HPA axis (Figure 1(a)). However, if these raises in glucocorticoid levels become too frequent, cells might develop “resistance to glucocorticoid action” [40, 48]. In fact, impaired negative feedback regulation of HPA axis function is a hallmark of major depression and is reflected by decreased responsiveness to glucocorticoids [40]. Of notice, resistance to glucocorticoids was also described in IL-10 producing cells [48]. The glucocorticoid resistance leads to a decrease in the production of IL-10 which might impact on the negative regulation of corticosterone production by the adrenal glands. In addition, decreased IL-10 can promote an imbalance in the cytokine milieu that would further activate the HPA axis (Figure 1(b)). This hypothesis fits well with the observations that, in certain circumstances, glucocorticoids trigger a proinflammatory action [49].
fig1

Figure 1: Schematic representation of the link between IL-10 and HPA axis. (a) in a basal situation and (b) during depression.

In the hypothesis outlined here we proposed IL-10 as an important link between the changes in hormonal and cytokine milieus that are of recognized relevance for depression. In the future, studies in molecules that might represent a link between the modulation of neural, endocrine, and immune systems are extremely important to further understand the etiology of depression. The cross-talk between these systems, mediated by IL-10, may become a target for novel antidepressant therapies.
Acknowledgments

The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for providing a fellowship to Roque S. (SFRH/BD/24539/2005). This work was supported by a Grant from FCT (PTDC/SAU-NEU/72699/2006).

"To summarize this first part of my talk, within the mucosa there is a cytokine milieu that is directed towards a pro-inflammatory response. Overall, there is evidence of a diffuse, subtle panenteric pathology, with increased inflammatory cell infiltrate. There is increased production of pro-inflammatory cytokines, and there is a decreased production of the regulatory cytokine IL10. There is also this possibility that an autoimmune response occurs that affects the epithelial cells and may lead to a disrupted intestinal barrier."